Biodegradable polyphosphates for controlled release of bioactive substances

ABSTRACT

The present invention is directed to a biodegradable system for the controlled release of bioactive substances. This system comprises novel biodegradable and biocompatible polyphosphoesters that carry positive charges. Process for making these polyphosphoesters, compositions containing these polyphosphoesters and biologically active substances, articles and methods for delivery of drugs and genes using this system are described. A controlled gene delivery system based on these polyphosphoesters is prepared by complex coacervation of nucleic acid (DNA or RNA) with polymers. The release rates can be manipulated by adjusting the charge ratios of polyphosphoesters to nucleic acids. This gene delivery system yields a higher gene expression in muscle when injected intramuscularly.

This application claims the benefit of U.S. Provisional Application Ser.No. 60/290,888 filed May 14, 2001, the teachings of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to biodegradable polymercompositions, in particular those containing both phosphoester linkagesin the polymer backbone and chargeable groups linked to the backbonethrough a phosphoester bond. These biodegradable polymers of theinvention are designed for the controlled release of drugs and genes,particularly as carriers for gene therapy and for the delivery ofprotein drugs. The invention also has wide applicability in tissueengineering applications, where the sustained delivery of growth factorsis achieved through gene transfer.

2. Background

Polymeric controlled drug delivery has significantly improved thesuccess of many drug therapies (Langer, 1990, New methods of drugdelivery, Science 249: 1527-33; Poznansky, et al., 1984, Biologicalapproaches to the controlled delivery of drugs: a critical review,Pharmacol. Rev. 36: 277-336). In such a delivery system,pharmacokinetics and biodistribution of the drug depend upon thephysiochemical properties and/or degradation properties of the polymercarriers. In general, polymeric carriers offer advantages over otherdelivery systems: polymeric systems potentially have more controllablerelease kinetics, better stability in storage, and have betterbiocompatibility. A biodegradable drug-carrier could offer featuresdifficult to attain from non-biodegradable systems. Other than obviatingthe need to remove the drug-depleted devices, a biodegradable system isalso applicable to a wider range of drugs. More and more new polymercarriers have been proposed for controlled drug delivery, althoughpoly(lactide-co-glycolide) copolymers still dominate the field. There isclearly justification to continue to develop new biodegradabledrug-carriers, because of the increasing need in the emerging newapplications. The widening scope of applications requires polymericcarriers to assume different configurations and serve additionalfunctions other than just passive delivery. For instance, applying thecontrolled release device as more than just a monolithic matrix, forexample, as a coating material for a drug-eluting stent, may obligatethe polymer to have elastomeric properties. In the new and excitingfield of tissue engineering where local and sustained delivery of growthfactors and/or genes encoding these growth factors may influence thecourse of tissue development, the drug-carrier may also need to performa double-duty to provide structural support or scaffolding functions. Toachieve active targeting, it would involve conjugation of ligands to thepolymeric carriers, which requires the polymeric carrier to containfunctional groups for derivatization. In the field of gene delivery,polymeric gene carriers need to be of polycationic nature and shouldhave the structural flexibility to include targeting feature andparameters affecting the intracellular trafficking of the genes (Han, etal., 2000, Development of biomaterials for gene therapy, MolecularTherapy 2: 302-317; Varga, et al., 2000, Receptor-mediated targeting ofgene delivery vectors: insights from molecular mechanisms for improvedvehicle design. Biotechnol. Bioeng. 70: 593-605). With such a broadutility for these biodegradable drug-carriers, no one single materialcan be expected to satisfy all requirements of different applications.

Gene therapy has been progressively developed with the hope that it willbe an integral part of medical modalities in the future. Gene deliverysystem is one of the key components in gene medicine, which directs thegene expression plasmids to the specific locations within the body. Thecontrol of gene expression is achieved by influencing the distributionand stability of plasmids in vivo and the access of the plasmids to thetarget cells, and affecting the intracellular trafficking steps of theplasmids (Mahato, et al., 1999, Pharmaceutical perspectives of nonviralgene therapy, Adv. Genet. 41: 95-156). Recently, there is an increasinginterest in developing systems for sustained release of DNA. Such asystem could be used to achieve localized and enhanced gene expressionin skeletal muscle. It would find wide applications in treating muscleand nerve disorders, providing systemic circulation of secretoryproteins, and as a genetic vaccine carrier. Encapsulation of DNA in PLGAnanoparticles (Cohen, et al., 2000, Sustained delivery and expression ofDNA encapsulated in polymeric nanoparticles. Gene Therapy 7: 1896-1905)and absorption of plasmid to the surface of cationic PLGA microparticles(Singh, et al., 2000, Characterization of cationic microparticles withadsorbed plasmid DNA. Proceed. Int'l. Symp. Control. Rel. Bioact.Mater., 27, 6405-6406) have been reported recently to achieve sustainedrelease of plasmid DNA. Sustained release of DNA was observed for 2 to 4weeks in these systems. The cationic microparticles induced aboutfour-fold higher gene expression level in muscle at day 14, and inducedhigher Th1 and Th2 responses in mice (Singh, et al., 2000, Cationicmicroparticles: A potent delivery system for DNA vaccines. Proc. Natl.Acad. Sci. USA 97(2): 811-816). However, both systems are limited by thelow DNA loading levels (<1%) and the little room for optimization of DNArelease kinetics. Other systems currently under investigations arenon-biodegradable polymeric systems, e.g. poly(ethylene-co-vinylacetate) (Luo, et al., 1999, Controlled DNA delivery systems. Pharm.Res. 16(8): 1300-1308) and Poloxamers (Lemieux, et al., 2000, Acombination of poloxamers increases gene expression of plasmid DNA inskeletal muscle. Gene Therapy 7: 986-991). The present patent features anovel gene delivery system that based on the biodegradation of polymericcarriers to achieve a sustained release of plasmid DNA in a controlledmanner.

SUMMARY OF THE INVENTION

The invention provides positively chargeable biodegradable polymers thatcomprises at least one phosphoester linkage in the polymer backbone andat least one positively chargeable group wherein the positivelychargeable group is a substitutent of a side chain attached to thepolymer backbone through a phosphoester linkage.

The invention further provides positively chargeable biodegradablepolymer compositions comprising:

(a) at least one biologically active substance; and

(b) A positively chargeable biodegradable polymer comprising at leastone phosphoester linkage in the polymer backbone and at least onepositively chargeable group wherein the positively chargeable group is asubstituent of a side chain attached to the polymer backbone through aphosphoester linkage.

The invention additionally provides a method of preparing a positivelychargeable biodegradable polymers. The method comprising the steps of:

polymerizing at least one monomer to form a polymer with at least onephosphoester linkage in the polymer backbone;

reacting the polymer with an alcohol having a positively chargeablegroup or a substituent that can be functionalized to a positivelychargeable group under conditions conducive to the formation of apositively chargeable biodegradable polymer comprising at least onephosphoester linkage in the polymer backbone and at least one positivelychargeable group wherein the positively chargeable group is asubstitutent of a side chain attached to the polymer backbone through aphosphoester linkage.

The invention provides a method of preparing a positively chargeablebiodegradable polymer composition. The method comprises the steps of:

providing a positively chargeable biodegradable polymer comprising atleast one phosphoester linkage in the polymer backbone and at least onepositively chargeable group wherein the positively chargeable group is asubstitutent of a side chain attached to the polymer backbone through aphosphoester linkage.

contacting the positively chargeable biodegradable polymer with abiologically active substance under conditions conducive to theformation of a complex comprising the positively chargeablebiodegradable polymer and the biologically active substance.

The invention also provides for the controlled release of a biologicallyactive substance in-vivo or in-vitro. The method comprises the steps of:

providing a positively chargeable biodegradable polymer compositioncomprising:

(a) at least one biologically active substance; and

(b) A positively chargeable biodegradable polymer comprising at leastone phosphoester linkage in the polymer backbone and at least onepositively chargeable group wherein the positively chargeable group is asubstituent of a side chain attached to the polymer backbone through aphosphoester linkage;

contacting the composition in vivo or in vitro with a biological fluid,cell or tissue under conditions conducive to the delivery of at least aportion of the biologically active substance to the biological fluid,cell or tissue.

The invention further provides methods for gene transfection using thecontrolled release methods and the positively chargeable biodegradablepolymer composition comprising a DNA sequence, a gene or a genefragment, to deliver a DNA sequence, a gene or a gene fragment to aspecified tissue target in a patient. Gene transfection methods of theinvention are suitable for use in treatment of any disease or disorderwhich is currently treatable by gene therapy or is contemplated as adisease or disorder suitable for treatment by gene therapy in the forfuture. Gene transfection methods of the invention comprise the steps of

providing a positively chargeable biodegradable polymer compositioncomprising:

(a) at least one biologically active substance; and

(b) A positively chargeable biodegradable polymer comprising at leastone phosphoester linkage in the polymer backbone and at least onepositively chargeable group wherein the positively chargeable group is asubstituent of a side chain attached to the polymer backbone through aphosphoester linkage;

contacting the composition with a biological fluid, cell or tissue underconditions conducive to the delivery of at least a portion of the DNAsequence, gene or gene fragment to the biological fluid, cell or tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Synthesis scheme of PPE-EA;

FIG. 2. Gel permeation chromatograph of PPE-EA;

FIG. 3. Cytotoxicity of PPE-EA in COS-7 Cells as compared with PEI andPLL;

FIG. 4. Gel electrophoretic analysis of the complexation of PPE-EA withDNA;

FIG. 5. In vitro release profiles of plasmid DNA from PPE-EA-DNAcoacervates prepared at different charge ratios;

FIG. 6. In vitro transfection efficiency of PPE-EA-DNA coacervates inHEK 293 cells;

FIG. 7. Beta-galactosidase level in mouse muscle following intramucularinjection of PPE-EA-DNA coacervates at a dose of 2 μg DNA/muscle;

FIG. 8. Tissue response at day 7 following intramuscular injection ofPPE-EA and PEI. (a) Saline injection (40 μl), showed normal tissue; (b)& (c): PPE-EA injection (13.1 μg, 60 nmole amino group), showed focalinflammation; (d) & (e): PEI (Mw 25 KDa, 2.5 μg, 60 nmole chargedgroups), showed severe inflammation and muscle necrosis; and

FIG. 9. IFN-α2b concentration in serum of mice following intramuscularinjection of naked DNA (n=5˜7) or complexes at N/P ratio of 0.5 (n=5˜7).Each mouse received a dose equivalent to 50 μg of plasmid DNA. Naïvemice served as a control (n=4).

DETAILED DESCRIPTION OF THE INVENTION

The biodegradable polyphosphoesters of the invention comprise therecurring monomeric units shown in the Formula I:

wherein:

R₁ is a divalent organic moiety that is aliphatic, aromatic orheterocyclic;

R₂ is alkyl, aryl, heteroaryl, heteroalicyclic, cycloalkyl, aralkyl, orcycloalkylalkyl; and

each occurrence of R₂ is substituted with one or more positivelychargeable functional groups (e.g. primary amino group, secondary aminogroup, tertiary amino group and quaternary amino group, etc.); and

n is 5 to 2000.

Particularly preferred polymers according to formula I include polymersof formula II:

wherein:

R₂ is as defined in Formula I:

R₃ and R₄ are independently selected at each occurrence of R₃ and R₄from the group consisting of hydrogen, alkyl, cycloalkyl, alkoxy, aryl,heteroaryl, heteroalicyclic, aralkyl, a steroid derivative; and

q is an integer from about 1 to about 5.

Preferred positively chargeable biodegradable polymers of the inventionare capable of forming a complex with negatively charged or neutralbiologically active substances. Preferred biologically active substancesinclude DNA, RNA, proteins, small molecule therapeutics, and the like.

Other preferred positively chargeable biodegradable polymers of theinvention include polymers capable of complexing 20-60% by weight of anegatively charged or neutral biologically active substance such as DNA,RNA, proteins, small molecule therapeutics, and the like.

Furthermore, preferred positively chargeable biodegradable polymers ofthe invention include polymers having between about 5 and about 2,000phosphate groups, more preferably between about 10 and about 1500phosphate groups, and particularly preferred are polymers having betweenabout 20 and 1000 phosphate groups. Also preferred are polymers having amolecular weight of between about 1000 and 500,000, more preferablyhaving a molecular weight of between about 2000 and 200,000,particularly preferable are polymers having a molecular weight ofbetween about 2000 and 100,000.

In additional preferred embodiments, positively chargeable biodegradablepolymers of the invention further comprise one or more groups thatfacilitate intracellular or extracellular delivery of a negativelycharged or neutral biologically active substance. Preferred groups forfacilitating intracellular delivery of a biologically active substanceinclude a lysosomalytic agent, an amphiphilic peptide, a steroidderivative, and the like.

In preferred embodiments, the biodegradable polyphosphoester polymers ofthe invention, including polymers according to Formula I or Formula II,are biocompatible before and upon degradation.

In another embodiment, the invention features a coacervate system usefulfor the delivery of bioactive macromolecules comprising thebiodegradable polymer of Formula I.

In a further embodiment, the invention contemplates a process of makingpolymeric coacervates for delivery of bioactive macromolecules.

In yet another embodiment, the invention comprises articles comprisingone or several different polymers with structures shown in Formula I andbioactive substances, e.g. nucleic acids and other negatively chargedmacromolecules for sustained release of these bioactive substancesin-vivo and in-vitro. Additionally, the bioactive substances can bereleased in a controlled, sustained manner either an intracellular andextracellular manner. In a still further embodiment, the inventioncontemplates a process for preparing biodegradable polyphosphoesters,which comprises a step of reacting a polymer shown in Formula III,wherein X is a halogen and R¹ is as defined in Formula I, with analcohol having a general structure as R²OH, wherein R₂ is alkyl, aryl,heteroaryl, heteroalicyclic, cycloalkyl, aralkyl or cycloalkylalkylwherein each occurrence of R₂ is substituted with one or more positivelychargeable functional groups (e.g., primary amino group, secondary aminogroup, tertiary amino group and quaternary amino group, etc.).

In specific embodiments, one or more charged groups present in R₂ arecapable of reacting with a P-halogen bond. Preferably, reactive chargedgroups are protected using standard organic chemistry protecting grouptechniques. The protected alcohol, R₂OH, is then reacted with thepolymer of Formula III. In particularly preferred embodiments, reactivecharged groups include primary or secondary amine groups.

The biodegradable polymeric system described in the present inventionachieves sustained and localized delivery of one or more therapeuticagents to a designated biological tissue or site in a patient. Inparticular, the polymeric system of the invention achieves sustained andlocalized delivery of one or more genes in skeletal muscles orintradermally and achieve a higher gene transfer efficiencies than otherplasmid delivery systems currently under investigation.

The polyphosphate carriers of the present invention typically offer thefollowing advantages over other biodegradable carriers described in theliterature and patents:

The polyphosphate carriers of the present invention are more efficientat binding to nucleic acids and proteins. In general, polymers providedherein have higher molecular weight than most other biodegradablecarriers and a relatively high charge density, which leads to anincreased binding capacity to plasmid DNA. Increased DNA bindingcapacity results in increased nucleic acid loading levels for thepolyphosphate carriers provided herein. Compared to the PLGAmicroparticle systems reported in the literatures, the coacervate systemprovided by the present invention is capable of much higher loadinglevels of nucleic acids (in a range of 20 to 60%, as compared with lessthan 1% for the microparticle systems reported in the literatures). Thisis particularly beneficial when a higher dose of administration isneeded.

The structures of the polyphosphate carriers of the present inventioncan be modified to have variable charge groups with different pKb,different charge density, molecular weight,hydrophilicity/hydrophobicity balance to optimize the degradation rateof the polymers, nucleic acid release rates from the systems andtransfection activity of the polymers. Sustained release of plasmid DNAis achieved either in an extracellular or intracellular manner. For theintracellular delivery and sustained release of nucleic acids, alysosomalytic agent, e.g. an amphiphilic peptide, could be conjugated tothe carriers to enhance the lysosomal escape after cell uptake. Alipophilic moiety, e.g. a group bearing cholesterol structural or lipid,could be conjugated to the carriers to enhance the interaction betweencoacervates and cell membrane therefore facilitate cell uptake. Anucleus localization signal could be conjugated to the carriers tofacilitate the nucleus translocation.

Polyphosphate polymers of the invention are biodegradable, such polymershave a cleavable backbone, which is cleaved by at least one pathwayselected from hydrolystic or enzymatic degradation.

Polyphosphate polymers of the invention are biocompatible before, duringand after biodegradation. Biodegradation breakdown products aretypically non-toxic. The polyphosphoramidate polymers of the inventionare less cytotoxic poly-L-lysine, PEI and liposome compositions invitro. In one of the embodiment, polymer of Formula I degrades tophosphate, 1,2-propanediol and ethanolamine. The cytotoxicity assaysuggests minimal toxicity when incubated with cells for 24 hours at aconcentration higher than 500 μg/ml.

Polyphosphates suitable for use in the invention may be modified tocomprise one or more specific ligands conjugated to the side chain or asa side chain group to enhance the cellular uptake or one or morebioactive molecules (nucleic acids and proteins) dispersed in carrierpolymer and/or achieve tissue/cell specific delivery of the bioactivecargo.

Polyphosphates suitable for use in the methods of the present inventioninclude any and all different single pure isomers and mixtures of two ormore isomers. The term isomer is intended to include diastereoisomers,enantiomers, regioisomers, structural isomers, rotational isomers,tautomers, and the like. For compounds which contain one or morestereogenic centers, e.g., chiral compounds, the methods of theinvention may be carried out with a enantiomerically enriched compound,a racemate, or a mixture of diastereomers. Preferred enantiomericallyenriched compounds have an enantiomeric excess of 50% or more, morepreferably the compound has an enantiomeric excess of 60%, 70%, 80%,90%, 95%, 98%, or 99% or more.

Polyphosphates suitable for use in the methods of the present inventioninclude any and all molecular weight distribution profiles, i.e.,polymers having a M_(w), or M_(n) of between 1 and about 50, moretypically a M_(w), or M_(n) between about 1.2 and about 10. Moreover,polyphosphroamidates of the invention have a polydispersity index ofbetween about 1 and about 5.

As also discussed above, typical subjects for administration inaccordance with the invention are mammals, such as primates, especiallyhumans.

Biodegradable polymers differ from non-biodegradable polymers in thatthey can be degraded during in vivo therapy. This generally involvesbreaking down the polymer into its monomeric subunits. In principle, theultimate hydrolytic breakdown products of polymers suitable for use inthe methods of the present invention should be biocompatible, non-toxicand easily excreted from a patient's body. However, the intermediateoligomeric products of the hydrolysis may have different properties.Thus, toxicology of a biodegradable polymer intended for implantation orinjection, even one synthesized from apparently innocuous monomericstructures, is typically determined after one or more toxicity analyses.

The biodegradable polymer of the invention is preferably sufficientlypure to be biocompatible itself and remains biocompatible uponbiodegradation. “Biocompatible” is defined to mean that thebiodegradation products and/or the polymer itself are nontoxic andresult in only minimal tissue irritation when instilled in the bladderor transported or otherwise localized to other tissues within a patient.

It will be appreciated that the actual preferred amounts of therapeuticagent or other component used in a given composition will vary accordingto the therapeutic agent being utilized including the polymer systembeing employed, the mode of application, the particular site ofadministration, etc. Optimal administration rates for a given protocolof administration can be readily ascertained by those skilled in the artusing conventional dosage determination tests conducted with regard tothe foregoing guidelines.

As used herein, “alkyl” is intended to include branched, straight-chainand cyclic saturated aliphatic hydrocarbon groups including alkylene,having the specified number of carbon atoms. Examples of alkyl include,but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl,s-butyl, i-butyl, n-pentyl, and s-pentyl. Alkyl groups typically have 1to about 16 carbon atoms, more typically 1 to about 20 or 1 to about 12carbon atoms. Preferred alkyl groups are C₁-C₂₀ alkyl groups, morepreferred are C₁₋₁₂-alkyl and C₁₋₆-alkyl groups. Especially preferredalkyl groups are methyl, ethyl, and propyl.

As used herein, “heteroalkyl” is intended to include branched,straight-chain and cyclic saturated aliphatic hydrocarbon groupsincluding alkylene, having the specified number of carbon atoms and atleast one heteroatom, e.g., N, O or S. Heteroalkyl groups will typicallyhave between about 1 and about 20 carbon atoms and about 1 to about 8heteroatoms, preferably about 1 to about 12 carbon atoms and about 1 toabout 4 heteroatoms. Preferred heteroalkyl groups include the followinggroups. Preferred alkylthio groups include those groups having one ormore thioether linkages and from 1 to about 12 carbon atoms, morepreferably from 1 to about 8 carbon atoms, and still more preferablyfrom 1 to about 6 carbon atoms. Alylthio groups having 1, 2, 3, of 4carbon atoms are particularly preferred. Prefered alkylsulfinyl groupsinclude those groups having one or more sulfoxide (SO) groups and from 1to about 12 carbon atoms, more preferably from 1 to about 8 carbonatoms, and still more preferably from 1 to about 6 carbon atoms.Alkylsulfinyl groups having 1, 2, 3, or 4 carbon atoms are particularlypreferred. Preferred alkylsulfonyl groups include those groups havingone or more sulfonyl (SO₂) groups and from 1 to about 12 carbon atoms,more preferably from 1 to about 8 carbon atoms, and still morepreferably from 1 to about 6 carbon atoms. Alylsulfonyl groups having 1,2, 3, or 4 carbon atoms are particularly preferred. Preferred aminoalkylgroups include those groups having one or more primary, secondary and/ortertiary amine groups, and from 1 to about 12 carbon atoms, morepreferably from 1 to about 8 carbon atoms, and still more preferablyfrom 1 to about 6 carbon atoms. Aminoalkyl groups having 1, 2, 3, or 4carbon atoms are particularly preferred.

As used herein, “heteroalkenyl” is intended to include branched,straight-chain and cyclic saturated aliphatic hydrocarbon groupsincluding alkenylene, having the specified number of carbon atoms and atleast one heteroatom, e.g., N, O or S. Heteroalkenyl groups willtypically have between about 1 and about 20 carbon atoms and about 1 toabout 8 heteroatoms, preferably about 1 to about 12 carbon atoms andabout 1 to about 4 heteroatoms. Preferred heteroalkenyl groups includethe following groups. Preferred alkylthio groups include those groupshaving one or more thioether linkages and from 1 to about 12 carbonatoms, more preferably from 1 to about 8 carbon atoms, and still morepreferably from 1 to about 6 carbon atoms. Alkenylthio groups having 1,2, 3, or 4 carbon atoms are particularly preferred. Preferedalkenylsulfinyl groups include those groups having one or more sulfoxide(SO) groups and from 1 to about 12 carbon atoms, more preferably from 1to about 8 carbon atoms, and still more preferably from 1 to about 6carbon atoms. Alkenylsulfinyl groups having 1, 2, 3, or 4 carbon atomsare particularly preferred. Preferred alkenylsulfonyl groups includethose groups having one or more sulfonyl (SO₂) groups and from 1 toabout 12 carbon atoms, more preferably from 1 to about 8 carbon atoms,and still more preferably from 1 to about 6 carbon atoms.Alkenylsulfonyl groups having 1, 2, 3, or 4 carbon atoms areparticularly preferred. Preferred aminoalkenyl groups include thosegroups having one or more primary, secondary and/or tertiary aminegroups, and from 1 to about 12 carbon atoms, more preferably from 1 toabout 8 carbon atoms, and still more preferably from 1 to about 6 carbonatoms. Aminoalkenyl groups having 1, 2, 3, or 4 carbon atoms areparticularly preferred.

As used herein, “heteroalkynyl” is intended to include branched,straight-chain and cyclic saturated aliphatic hydrocarbon groupsincluding alkynylene, having the specified number of carbon atoms and atleast one heteroatom, e.g., N, O or S. Heteroalkynyl groups willtypically have between about 1 and about 20 carbon atoms and about 1 toabout 8 heteroatoms, preferably about 1 to about 12 carbon atoms andabout 1 to about 4 heteroatoms. Preferred heteroalkynyl groups includethe following groups. Preferred alkynylthio groups include those groupshaving one or more thioether linkages and from 1 to about 12 carbonatoms, more preferably from 1 to about 8 carbon atoms, and still morepreferably from 1 to about 6 carbon atoms. Alkynylthio groups having 1,2, 3, or 4 carbon atoms are particularly preferred. Preferedalkynylsulfinyl groups include those groups having one or more sulfoxide(SO) groups and from 1 to about 12 carbon atoms, more preferably from 1to about 8 carbon atoms, and still more preferably from 1 to about 6carbon atoms. Alkynylsulfinyl groups having 1, 2, 3, or 4 carbon atomsare particularly preferred. Preferred alkynylsulfonyl groups includethose groups having one or more sulfonyl (SO₂) groups and from 1 toabout 12 carbon atoms, more preferably from 1 to about 8 carbon atoms,and still more preferably from 1 to about 6 carbon atoms,Alkynylsulfonyl groups having 1, 2, 3, or 4 carbon atoms areparticularly preferred. Preferred aminoalkynyl groups include thosegroups having one or more primary, secondary and/or tertiary aminegroups, and from 1 to about 12 carbon atoms, more preferably from 1 toabout 8 carbon atoms, and still more preferably from 1 to about 6 carbonatoms. Aminoalkynyl groups having 1, 2, 3, or 4 carbon atoms areparticularly preferred.

As used herein, “cycloalkyl” is intended to include saturated ringgroups, having, the specified number of carbon atoms, such ascyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. Cycloalkyl groupstypically will have 3 to about 8 ring members.

In the term “(C₃₋₆ cycloalkyl)C₁-₄ alkyl”, as defined above, the pointof attachment is on the alkyl group. This term encompasses, but is notlimited to, cyclopropylmethyl, cyclohexylmethyl, cyclohexylmethyl.

As used here, “alkenyl” is intended to include hydrocarbon chains ofstraight, cyclic or branched configuration, including alkenylene, andone or more unsaturated carbon-carbon bonds which may occur in anystable point along the chain, such as ethenyl and propenyl. Alkenylgroups typically will have 2 to about 12 carbon atoms, more typically 2to about 12 carbon atoms.

As used herein, “alkynyl” is intended to include hydrocarbon chains ofstraight, cyclic or branched configuration, including alkynylene, andone or more triple carbon-carbon bonds which may occur in any stablepoint along the chain, such as ethynyl and propynyl. Alkynyl groupstypically will have 2 to about 20 carbon atoms, more typically 2 toabout 12 carbon atoms.

As used herein, “haloalkyl” is intended to include both branched andstraight-chain saturated aliphatic hydrocarbon groups having thespecified number of carbon atoms, substituted with 1 or more halogen(for example —C_(v)F_(W) where v=1 to 3 and w=1 to (2v+1). Examples ofhaloalkyl include, but are not limited to, trifluoromethyl,trichloromethyl, pentafluoroethyl, and pentachloroethyl. Typicalhaloalkyl groups will have 1 to about 16 carbon atoms, more typically 1to about 12 carbon atoms.

As used herein, “alkoxy” represents an alkyl group as defined above withthe indicated number of carbon atoms attached through an oxygen bridge.Examples of alkoxy include, but are not limited to, methoxy, ethoxy,n-propoxy, i-propoxy, n-butoxy, 2-butoxy, t-butoxy, n-pentoxy,2-pentoxy, 3-pentoxy, isopentoxy, neopentoxy, n-hexoxy, 2-hexoxy,3-hexoxy, and 3-methylpentoxy. Alkoxy groups typically have 1 to about16 carbon atoms, more typically 1 to about 12 carbon atoms.

“Prodrugs” are intended to include any covalently bonded carriers whichrelease the active parent drug according to formula I in vivo when suchprodrug is administered to a mammalian subject. Prodrugs of a compoundare prepared by modifying functional groups present in the drug compoundin such a way that the modifications are cleaved, either in routinemanipulation or in vivo, to the parent compound.

Combinations of substituents and/or variables are permissible only ifsuch combinations result in stable compounds. A stable compound orstable structure is meant to imply a compound that is sufficientlyrobust to survive isolation to a useful degree of purity from a reactionmixture, and formulation into an effective therapeutic agent.

As used herein, the term “aliphatic” refers to a linear, branched,cyclic alkane, alkene, or alkyne. Preferred aliphatic groups in thepoly(phosphoester-co-amide) polymer of the invention are linear orbranched and have from 1 to 20 carbon atoms.

As used herein, the term “aryl” refers to an unsaturated cyclic carboncompound with 4n+2 electrons where n is a non-negative integer, about5-18 aromatic ring atoms and about 1 to about 3 aromatic rings.

As used herein, the term “heterocyclic” refers to a saturated orunsaturated ring compound having one or more atoms other than carbon inthe ring, for example, nitrogen, oxygen or sulfur.

The polymers of the invention are usually characterized by a releaserate of the therapeutic agent in vivo that is controlled at least inpart as a function of hydrolysis of the phosphoester bond of the polymerduring biodegradation. Additionally, the therapeutic agent to bereleased may be conjugated to the sidechain of the phosphramidate repeatunit to form a pendant drug delivery system. Further, other factors arealso important.

The life of a biodegradable polymer in vivo also depends upon itsmolecular weight, crystallinity, biostability, and the degree ofcross-linking. In general, the greater the molecular weight, the higherthe degree of crystallinity, and the greater the biostability, theslower biodegradation will be.

The therapeutic agent of the invention can vary widely with the purposefor the composition. The agnet(s) may be described as a single entity ora combination of entities. The delivery system is designed to be usedwith therapeutic agents having high water-solubility as well as withthose having low water-solubility to produce a delivery system that hascontrolled release rates. The terms “therapeutic agent” and“biologically active substance” include without limitation, medicaments;vitamins; mineral supplements; substances used for the treatment,prevention, diagnosis, cure or mitigation of disease or illness; orsubstances which affect the structure or function of the body; orpro-drugs, which become biologically active or more active after theyhave been placed in a predetermined physiological environment.

Non-limiting examples of useful therapeutic agents and biologicallyactive substances include the following expanded therapeutic categories:anabolic agents, antacids, anti-asthmatic agents, anti-cholesterolemicand anti-lipid agents, anti-coagulants, anti-convulsants,anti-diarrheals, anti-emetics, anti-infective agents, anti-inflammatoryagents, anti-manic agents, anti-nauseants, anti-neoplastic agents,anti-obesity agents, anti-pyretic and analgesic agents, anti-spasmodicagents, anti-thrombotic agents, anti-uricemic agents, anti-anginalagents, antihistamines, anti-tussives, appetite suppressants,biologicals, cerebral dilators, coronary dilators, decongestants,diuretics, diagnostic agents, erythropoietic agents, expectorants,gastrointestinal sedatives, hyperglycemic agents, hypnotics,hypoglycemic agents, ion exchange resins, laxatives, mineralsupplements, mucolytic agents, neuromuscular drugs, peripheralvasodilators, psychotropics, sedatives, stimulants, thyroid andanti-thyroid agents, uterine relaxants, vitamins, antigenic materials,and prodrugs.

Specific examples of useful therapeutic agents and biologically activesubstances, i.e., bioactive molecules, from the above categoriesinclude: (a) anti-neoplastics such as androgen inhibitors,antimetabolites, cytotoxic agents, immunomodulators; (b) anti-tussivessuch as dextromethorphan, dextromethorphan hydrobromide, noscapine,carbetapentane citrate, and chlophedianol hydrochloride; (c)antihistamines such as chlorpheniramine maleate, phenindamine tartrate,pyrilamine maleate, doxylamine succinate, and phenyltoloxamine citrate;(d) decongestants such as phenylephrine hydrochloride,phenylpropanolamine hydrochloride, pseudoephedrine hydrochloride, andephedrine; (e) various alkaloids such as codeine phosphate, codeinesulfate and morphine; (f) mineral supplements such as potassiumchloride, zinc chloride, calcium carbonates, magnesium oxide, and otheralkali metal and alkaline earth metal salts; (g) ion exchange resinssuch as cholestryramine; (h) anti-arrhythmics such asN-acetylprocainamide; (i) antipyretics and analgesics such asacetaminophen, aspirin and ibuprofen; (j) appetite suppressants such asphenyl-propanolamine hydrochloride or caffeine; (k) expectorants such asguaifenesin; (l) antacids such as aluminum hydroxide and magnesiumhydroxide; (m) biologicals such as peptides, polypeptides, proteins andamino acids, hormones, interferons or cytokines and other bioactivepeptidic compounds, such as hGH, tPA, calcitonin, ANF, EPO and insulin;(n) anti-infective agents such as anti-fungals, anti-virals, antisepticsand antibiotics; and (o) antigenic materials, partricularly those usefulin vaccine applications.

Preferably, the therapeutic agent or biologically active substance isselected from the group consisting of DNA, polysaccharides, growthfactors, hormones, anti-angiogenesis factors, interferons or cytokines,and pro-drugs. In a particularly preferred embodiment, the therapeuticagent is a DNA vaccine comprising a DNA sequence encoding an antigen, aDNA sequence encoding a cytokine or a mixture of DNA sequences encodingan antigen and a cytokine.

Other preferred therapeutic agents are substances that are capable ofmodulating the immune response of a patient. Preferred therapeuticagents capable of modulating an immune response include protein vaccinesor DNA vaccines. More preferred therapeutic agents capable of modulatingan immune response are DNA vaccines. In general, DNA vaccine includevaccines which comprise a DNA sequence encoding an antigen, DNA sequenceencoding a cytokine or a combination of DNA sequence encoding an antigenand DNA sequence encoding a cytokine.

Preferred cytokine additives suitable for use in a DNA vaccine includecytokines selected from interleukins or interferons which can shift apatient's immune response toward either a T_(H)1 or T_(H)2 response.Preferred cytokines suitable for use in modulating an immune responseinclude interleukin-12, interleukin-10, interleukin-5, interleukin-4 andinterferon-gamma. Other preferred cytokines include interferon for usein treatment of Hepatitis C.

Suitable genes for use in the methods of the invention encodetherapeutic proteins for administration locally such as for use intreatment of muscle related diseases, such as the neuromusculardisorders and also for systemic delivery of therapeutic proteins such assecretory therapeutic proteins including, for example, interferon foruse in treatment of Hepatitis C.

Methods of the present invention are suitable for any protein or DNAbased vaccination method which induces either T_(H)1 T_(H)2 or acombination of T_(H)1 and T_(H)2 immunological responses. Methods areapplicable for any illness or disease for which a vaccination is knownor for which treatment is effected by systemic delivery of a therapeuticagent such as a small molecule drug, protein or DNA.

In preferred immune modulation methods of the invention, administrationmethods of the invention for delivering a therapeutic agent to the lymphnodes are suitable for delivering an therapeutic agent capable ofmodulating an immune response so that a patient's immune response ismodulated. The lymph node is one of the primary sites for immune systemstimulation; delivery of drugs, proteins, or DNA to these nodes resultsin the modulation of the immune response. After the microparticles areadministered to the lymph nodes, they deliver their the therapeuticagent contained therein. Release of the therapeutic can be observed inby direct visualization of protein expression in cells of the lymphnodes, as well as indirect evidence through positive immune responses.

Other preferred therapeutic agents are substances that are capable ofmodulating the immune response of a patient. Preferred therapeuticagents capable of modulating an immune response include protein vaccinesor DNA vaccines. More preferred therapeutic agents capable of modulatingan immune response are DNA vaccines. In general, DNA vaccine includevaccines which comprise a DNA sequence encoding an antigen, DNA sequenceencoding a cytokine or a combination of DNA sequence encoding an antigenand DNA sequence encoding a cytokine.

The therapeutic agents are used in amounts that are therapeuticallyeffective. While the effective amount of a therapeutic agent will dependon the particular material being used, amounts of the therapeutic agentfrom about 1% to about 65% have been easily incorporated into thepresent delivery systems while achieving controlled release. Lesseramounts may be used to achieve efficacious levels of treatment forcertain therapeutic agents.

In addition, the polymer composition of the invention can also comprisepolymer blends of the polymer of the invention with other biocompatiblepolymers, so long as they do not interfere undesirably with thebiodegradable characteristics of the composition. Blends of the polymerof the invention with such other polymers may offer even greaterflexibility in designing the precise release profile desired fortargeted drug delivery or the precise rate of biodegradability desiredfor structural implants such as for orthopedic applications. Examples ofsuch additional biocompatible polymers include other polycarbonates;polyesters; polyorthoesters; polyamides; polyurethanes;poly(iminocarbonates); and polyanhydrides.

As a drug delivery device, the polymer compositions of the inventionprovide a polymeric matrix capable of sequestering a biologically activesubstance and provide predictable, controlled delivery of the substance.The polymeric matrix then degrades to non-toxic residues.

It will be understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, and rate of excretion, drug combination (i.e., otherdrugs being administered to the patient), the severity of the particulardisease undergoing therapy, and other factors, including the judgment ofthe prescribing medical practitioner.

A positively chargeable biodegradable polymer composition of theinvention also may be packaged together with instructions (i.e. written,such as a written sheet) for treatment of a disorder as disclosedherein, e.g. instruction for treatment of a subject that is susceptibleto or suffering from a disease or disorder which may be treated byadministration of a bioactive molecule, e.g., therapeutic agent,dispersed in the positively chargeable biodegradable polymercomposition.

A positively chargeable biodegradable polymer composition of theinvention be administered parenterally, preferably in a sterilenon-toxic, pyrogen-free medium. The drug, depending on the vehicle andconcentration used, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anesthetics, preservatives andbuffering agents can be dissolved in the vehicle. The term parenteral asused herein includes injections and the like, such as subcutaneous,intradermal, intravascular (e.g., intravenous), intramuscular,intrasternal, spinal, intrathecal, and like injection or infusiontechniques, with subcutaneous, intramuscular and intravascularinjections or infusions being preferred.

A positively chargable biodegradable polymer composition of theinvention also may be packaged together with instructions (i.e. written,such as a written sheet) for treatment of a disorder as disclosedherein, e.g. instruction for treatment of a subject that is susceptibleto or suffering from inflammation, cellular injury disorders, or immunesystem disorders.

The present invention provides biocompatible and biodegradable genecarriers that release the DNA to a targeted tissue, cell or fluid in asustained manner. In one embodiment, the invention provides a PPE-EApolyphosphate as a gene carrier. The structure of PPE-EA is built on aphosphate backbone with a 1,2-propylene diol moiety, and a chargedaminoethyl side chain. The ultimate hydrolytic degradation products ofthis polymer are expected to be α-propylene glycol, phosphate andethanolamine, all with minimal toxicity profiles. Such a design has ledto a minimal toxicity profile of this polymer carrier, as demonstratedin the vigorous cytotoxicity assay, where gene carriers were incubatedwith cells for 24 hours. The acute tissue response to PPE-EA evaluatedin mouse muscles also revealed a similar contrast, when compared withPEI at a same dose of positive charges.

PPE-EA has a relatively higher molecular weight compared with most otherbiodegradable gene carriers known in the art. The relatively highmolecular weight of PPE-EA ( Mw: 30,300, degree of polymerization:100.2) was achieved by ring opening polymerization of a cyclic phosphatemonomer. This feature coupled with the pedent chain charges hasincreased the ability of PPE-EA to form condensate complexes with DNA.Complete binding of DNA was achieved with PPE-EA at a charge ratio of 1,which was several folds lower than other biodegradable gene carriers. Atthis charge ratio, PPE-EA was shown to effectively protect plasmid DNAfrom DNase I degradation and serum degradation.

Applicants have also surprisingly discovered that the relatively highdegradability of PPE-EA is suitable for the sustained release of DNAwhich occurs in connection with degradation of the polymeric phosphatecarrier. DNA release occurred as early as a few hours at the low N/Pratios, to a few days of retardation for high N/P ratios. N/P ratio wasthe factor that dominated the kinetics of DNA release, especially in thefirst phase (within the first 6 days), during which period the DNArelease rate decreased with the increase of N/P ratio. Such anadjustable DNA release kinetics is potentially advantageous in achievingdifferent intracellular and extracellular sustained release of DNA.Nevertheless, the DNA release rate should be optimized for eachparticular application.

The PPE-EA/DNA complexes of the invention offer superior controlledrelease profiles when compared with the biodegradable polymer basedsystems described in the literature, e.g. PLGA and polyanhydridemicro/nanoparticles. Loading levels of DNA achieved in this system weremuch higher than that in PLGA microparticles. For example, PPE-EA/DNAcomplexes with a N/P ratio of 1 have a DNA payload of 60%, comparingwith less than 2% for PLGA or polyanhydride microspheres. Higher loadinglevel reduced the use of carriers significantly. Moreover, thePPE-EA/DNA system was prepared by complex coacervation versus the doubleemulsion method for the microsphere systems. The former involved onlyaqueous conditions at room temperature, whereas the latter involvedorganic solvent and sonication/vortexing. The mild preparationconditions yielded good structural and functional integrity of the DNAreleased from PPE-EA/DNA complexes.

The sustained gene delivery system provided by the present invention issuitable for use in administration of gene delivery intramuscularly.This has to be compared to naked DNA injection, which is somewhateffective in affording both local and systemic transgene expression.Intramuscular administration of PPE-EA/DNA complexes resulted insignificantly higher and delayed β-gal expression in muscle, althoughthe enhancement effect becomes less prominent at higher administrationdose. The higher β-gal level in the PPE-EA mediated gene transfer ispresumably due to the sustained release of plasmid at the injection siteand the protection of plasmid by PPE-EA. Compositions of a positivelychargeable phosphate polymer and DNA provided by the present inventionwhich have a lower N/P ratio (<1) achieved higher levels of geneexpression, suggesting the complicated mechanism in gene delivery toskeletal muscle. Compositions having higher N/P ratios such as 1.5 or 2gave background level of gene expression in the muscle. This diminishedgene expression was not likely due to any toxicity of PPE-EA in thecomplexes at higher N/P ratio, since the total amount of PPE-EA used inthis formulation was only 20% of the dose tested for the tissuecompatibility experiment. Muscle biopsy in these groups did not show anynoticeable toxicity either. This is also consistent with the resultsfrom other cationic polymer/DNA complexes in muscle injectionformulation, where no positive gene transfection has been reported. Arecent study revealed that intramuscular injection of PEI/DNA complexesinduces gene expression in central nervous system (e.g. brain stem)through retrograde transport of the complexes/particles (13). It isunclear yet if these PPE-EA/DNA complexes were transported out of themuscle in a similar manner.

This enhancement of gene expression may be applied to delivertherapeutic proteins to systemic circulation such as the delivery ofIFN-α2b. This is a secretory protein that has a serum half-life of 1.7hours (14). An N/P ratio of 0.5 has been selected due to its higherefficiency in the previous study. Following intramuscular injection,PPE-EA/DNA complexes generated 1.8 times higher IFN-α2b in bloodcirculation than naked DNA on day 14 (p<0.05), although at other timepoints, the expression levels were similar to that of naked DNAinjection. This is particularly significant because of the shorthalf-life of IFN-α2b. It is worth noting that this result was obtainedat a relatively higher dose of DNA (50 μg). As suggested by the β-galexperiment, the enhancement effect by PPE-EA/DNA complexes might be moresignificant at lower doses.

The positively chargeable biodegradable polyphosphates provided by thepresent invention are the first polymeric carriers capable of inducinghigher levels of gene expression in muscle than naked DNA. Although theexperimental protocol was far from optimized, these results suggestedthe potential of PPE-EA as a gene carrier for the local delivery as wellas systemic delivery of protein pharmaceutics.

In one embodiment, the present invention provides a novel controlledgene delivery system based on a water soluble and biodegradablepolyphosphoester, poly(2-aminoethyl propylene phosphate) [PPE-EA]. Thepolymer degraded in PBS at 37° C. through the cleavage of the backbonephosphate bonds, and it was synthesized with a relative high molecularweight to ensure a suitable hydrolytic stability as a gene carrier. Thetissue response and cytotoxicity study demonstrated a better tissuecompatibility of PPE-EA in mouse muscle compared to commonly usedpolyethylenimine and poly-L-lysine. PPE-EA condensed DNA efficiently andprotected DNA from nuclease and serum degradation. Sustained release ofplasmid was achieved from PPE-EA/DNA complexes as a result of PPE-EAdegradation. The DNA release profiles appear to be predominantlycontrolled by carrier degradation and the release rate of plasmid couldbe adjusted by varying the charge ratio of PPE-EA to DNA. At an N/P(amino to phosphate groups) ratio of 1, a 46% burst was observed for thefirst day, followed by about 4% release per day (24 μg DNA/day/mg ofcomplex) for 12 days. Higher charge ratios reduced both the DNA releaserate and the burst effect. The released DNA retained its structural andfunctional integrity. Intramuscular injection of PPE-EA-p43-LacZcomplexes at N/P ratios of 0.5 and 1 resulted in enhancedβ-galactosidase expression in anterior Tibialis muscle in Balb/c mice,as compared with naked DNA injections. Similarly, PPE-EA/IFNα2b DNAcomplexes generated an increased systemic level of interferon-α2b inmouse serum following intramuscular injection, as compared with nakedDNA injection.

The following examples are illustrative of the invention. All documentsmentioned herein are incorporated herein by reference.

EXAMPLES

The following examples are offered by way of illustration and are notintended to limit the invention in any manner.

Example 1 Synthesis and Characterization of Polyphosphoramidates

The synthetic scheme is shown in FIG. 1.

1.1 Synthesis of poly(4-methyl-2-oxo-2-hydro-1,3,2-dioxaphospholane)

1.2

4-methyl-2-oxo-2-hydro-1,3,2-dioxaphospholane (58 g, 0.475 mol) [freshlyprepared according to Lucas' method (Lucas, Mitchell, and Scully, 1950,Cyclic phosphites of some aliphatic glycols. J. Am. Chem. Soc. 72:5491-5497) was polymerized in 200 ml of fresh dried CHCl₃ at roomtemperature for 48 hours. Polymerization was initiated withtriisobutylaluminum (1 wt %, 4 ml of 15% solution in heptane). Thepolymer was obtained by precipitation into anhydrous benzene. Thispolymer became insoluble in chloroform after precipitation, but it issoluble in anhydrous DMF.

1.2 Synthesis of poly(4-methyl-2-oxo-2-chloro-1,3,2-dioxaphospholane)

40 mL of polymer solution from the former step was added into 200 ml ofdried benzene. The precipitate was dried under vacuum and weighted toobtain white polymer (4.187 g). This polymer was then suspended in driedCH₂Cl₂ and dry Cl₂ was passed. The suspension was dissolved duringchlorination. Addition of Cl₂ was stopped at the first appearance of apersistent yellow color. The excess of Cl₂ was removed under vacuumuntil a colorless solution resulted. This solution was further used forpreparation of different derivatives without polymer isolation.

1.3 Synthesis ofPoly(4-methyl-2-oxo-2-(N-benzyloxycarbonyl)-aminoethyloxy-1,3,2-dioxaphospholane)

To the cooled solution ofpoly(4-methyl-2-oxo-2-chloro-1,3,2-dioxaphospholane) (24.4 mmol of P—Cl)in CH₂Cl₂ from the former step, was added DMAP (6.56 g, 53.6 mmol), thenthe solution of benzyl N-(2-hydroxyethyl) carbamate (5.24 g, 26.9 mmol)was added dropwise from an addition funnel. After the addition, themixture was heated and the mixture was allowed to reflux for 48 hours.The solution was washed with 1N HCl twice and water twice. The organiclayer was dried over anhydrous magnesium sulfate and concentrated. Theproduct was obtained by pouring the concentrated solution into ether asa white glassy solid (4.6 g, yield 60%).

1.4 Synthesis ofpoly(4-methyl-2-oxo-2-aminoethyloxy-1,3,2-dioxaphospholane)

Remove of the Cbz group was accomplished using formic acid and Pd/Caccording to a reported method (Zhou and Kohn, 1990, Preparation ofpoly(L-serine ester): A Structural analogue of conventionalpoly(L-serine), Macromolecules 23: 3399˜3406). In a 25 ml round-bottomflask the polymer (300 mg) was dissolved in 4 ml of DMF. To thissolution under a N₂ atmosphere was added 1 g of Pd/C (10%, Aldrich).With vigorous stirring formic acid (14 ml) was added dropwise over 15min. The reaction was stirred at room temperature for 14 hours andfiltered to remove Pd/C. The catalyst was washed with 20 ml of 1N HCl.The filtrates were combined and concentrated under vacuum using a waterbath below 45° C. to a volume of 5 ml whereupon 10 ml of 1N HCl wasadded. The solution was reconcentrated as above to a volume of 1 ml andthen added dropwise to 150 ml of acetone with stirring and cooled to−20° C. The polymer was isolated and dried thoroughly under vacuum.Yield 166 mg (80%). The structure of PPE-EA was confirmed by NMR. Theweight average molecular weight of PPE was 38,800 with a polydispersityindex of 1.64 as determined by GPC/LS/RI method (FIG. 2).

Example 2 Assay for the Cytotoxicity of PPE-EA

Cytotoxicity of PPE-EA in comparison with other potential gene carriers[poly-L-lysine (PLL) and polyethylenimine (PEI)] is evaluated using theMTT assay. COS-7 cells (6,000 per well) were seeded in 96-well platesand incubated for 24 hours at 37° C. in 5% CO₂ followed by addingpolymer solutions (50 μl) at different concentrations (0-1 mg/ml). After24 hours incubation, cell viability was analyzed by a MTT assayaccording to Hansen's method (Hansen, et al., 1989, J. Immunol. Methods,119: 203-210).

The assay results showed no significant change in morphology andproliferation rate as compared with cells without treatment, when PPE-EAwas incubated with cells for 24 hours at a dose up to 0.5 mg/ml. Incontrast, LD₅₀ values of PEI and PLL in this assay were below 10 μg/ml(FIG. 3). Similar results were observed in HEK 293 cells. This suggestedthat PPE-EA have a minimal cytotoxicity.

Example 3 Gel Retardation Assay for the DNA Binding Capacity of PPE-EA

To 2 μg or plasmid DNA dissolved in 20 μl of saline was added a PPE-EAsolution in 20 μl of saline at the increasing charge ratios from 0.5 to10 respectively. The mixture was vortexed for 20 seconds and thecoacervates were incubated at room temperature for 30 minutes and then10 μl of coacervates was analyzed on a 0.8% agrose gel. Formation ofcoacervates between plasmid DNA and PPE-EA was confirmed by gelretardation assay (FIG. 4). Complete binding of plasmid DNA was achievedat a charge ratio (N/P ratio) of 1 and above. PPE-DNA coacervates atcharge ratio of 1 and above provided partial protection to plasmid DNAfrom nuclease degradation.

Example 4 Preparation of Coacervates and Release of Plasmid DNA FromPPE-DNA Coacervates

PPE-EA-DNA coacervates were prepared in PBS with 1 mM EDTA by mixingplasmid DNA (60 μg/ml) with PPE-EA solution with concentration rangingfrom 20 μg/ml to 80 μg/ml to achieve various charge ratios (0.5 to 2).The coacervates were incubated at room temperature for 30 minutes beforeuse. Plasmid DNA release from the coacervates was performed at 37° C. Atvarious time points, samples were centrifuged and DNA concentration inthe supernatant was measured by UV spectrophotometry at 260 nm. Theintegrity of DNA released form the coacervates was analyzed by gelelectrophoresis (0.8% agrose gel).

Plasmid DNA released from PPE-EA-DNA coacervates over a period of twoweeks when the coacervates were incubated in PBS. A sustained releaseprofile for up to two weeks from the coacervates was observed as aresult of degradation of PPE. The release rate was a function of chargeratio (FIG. 5). When the coacervates were prepared at a N/P ratio of 1,a burst of 14.1 μg/ml was observed for the first day, then followed by anear constant release of 1.24 μg/ml/day for 12 days. At a N/P ratio of1.5, DNA release followed first order release for the first week andthen was near constant for the next week at a rate of 1.1 μg/ml/day.Whereas at a charge ratio of 2, DNA released at a slower rate, with anaverage of 3.2 μg/ml/day for the first 4 days and then 0.8 μg/ml/day forthe next nine days. No burst effect was observed for N/P ratios of 1.5and above. DNA release from these PPE-EA-DNA coacervates remained intactas indicated by the gel electrophoretic analysis, although some nickingof plasmid occurred during the incubation.

Example 5 Transfection Efficiency of PPE-EA-DNA Complex in HEK293 Cells

In vitro transfection of HEK293 cells with PPE-EA-DNA coacervates wasevaluated using luciferase as a marker gene. Cells were seeded 24 hoursprior to transfection into a 24-well plate (Becton-Dickinson, LincolnPark, N.J.) at a density of 8×10⁴ per well with 1 ml of complete medium(DMEM containing 10% FBS, supplemented with 2 mM L-glutamate, 50units/ml penicillin and 50 μg/ml streptomycin). At the time oftransfection, the medium in each well was replaced with 1 ml of serumfree DMEM. PPE-EA-DNA coacervates or PEI-DNA complexes or PLL-DNAcomplexes were incubated with the cells for 3 hours at 37° C. The mediumwas replaced with 1 ml of fresh complete medium and cells were furtherincubated for 48 hours. All the transfection tests were performed intriplicate. After the incubation, cells were permeabilized with 200 μlof cell lysis buffer (Promega Co., Madison, Wis.). The luciferaseactivity in cell extracts was measured using a luciferase assay kit(Promega Co., Madison, Wis.) on a luminometer (Lumat9605, EG&G Wallac).The light units (LU) were normalized against protein concentration inthe cell extracts, which was measured using BCA protein assay kit(Pierce, Rockford, Ill.).

FIG. 6 showed the transfection efficiency of PPE-EA-DNA coacervatesprepared at different charge ratios. As the gel electrophoresis analysisshowed, at a +/− charge ratio of 1.0 and above, all the plasmid DNAadded to the preparation mixture complexed with PPE-EA. However,coacervates with a charge ratio lower than 4 failed to show significantgene expression level. The highest level of gene transfection wasobserved with the coacervates synthesized at +/− charge ratios between 6and 8.

The transfection ability of PPE-EA-DNA coacervates suggested that thecontrolled release feature of this gene delivery technology could alsobeneficial in an intracellular gene delivery application.

Chloroquine was shown to enhance the transfection efficiency ofPPE-EA-DNA coacervates in vitro. FIG. 6 also included the transfectionefficiency of PPE-EA-DNA coacervates with 100 μg/ml of CQ as acomparison. It was evident that CQ can enhance the transfectionefficiency for about 10 to 100 times at this concentration. Theenhancement effect was more prominent for the coacervates prepared atlower charge ratio, suggesting the competition between the endosomalescape and DNA release and degradation inside the cells.

Example 6 β-Galactosidase Expression in Mouse Muscle FollowingIntramuscular Injection

Balb/c mice (three per group) received bilateral injections in theanterior tibialis muscle of 2 μg of p43-LacZ, or PPE-p43-LacZcoacervates prepared in saline at different charge ratios (0.5 and 1.0).The injected muscles were isolated on day 1, 3, 7 and 14. The expressionlevel of β-galactosidase in the muscle was measured using a β-GalReporter Gene Assay kit (Roche Molecular Biochemicals) usingβ-galactosidase as a standard.

Intramuscular injection of PPE-p43-LacZ complexes at an N/P ratio of 0.5resulted in a sustained level of β-galactosidase expression in anteriortibialis muscle in Balb/c mice. At a dose of 2 μg of DNA per muscle,PPE-DNA complexes with an N/P ratio of 0.5 yielded a 20-fold higherβ-Gal expression on day 7 than naked DNA group and the gene expressionlevel persisted for upto 4 weeks (FIG. 7). Interestingly, complexes withan N/P ratio of 1, although had a delayed gene expression than naked DNAinjection, yielded a lower level of β-Gal expression than complexes atN/P ratio of 0.5. A different set of experiment with a dose of 10 μg ofDNA injection showed the similar trend.

Example 7 Tissue Response of PPE-EA in Mouse Muscle

PPE-EA or PEI dissolved in saline (40 μl) was injected into the tibialisanterior muscle in Balb/c mice at a dose equivalent to 60 nmol ofpositive charge (13.1 μg/40 μl for PPE-EA and 2.5 μg/40 μl for PEI). Themuscles received the polymer injections were isolated at days 7, fixedin phosphate buffered formalin (10%), washed, and embedded in paraffin.Tissue sections were cut with 8 μm in thickness, placed on gelatincoated slides, and stained with hematoxylin and eosin (H&E) forhistological examination. The tissue response was evaluated by anindependent pathologist. Mice receiving intramuscular injection of 40 μlof saline were used as a control for this experiment.

Six to eight-week-old female Balb/c mice were obtained and housed inNational University of Singapore Animal Holding Unit. Mice weremaintained on ad libitum rodent feed and water at room temperature, 40%humidity. All animal procedures were approved by the National Universityof Singapore Faculty of Medicine Animal Care and Use Committee.

The acute tissue response to PPE-EA was evaluated in muscles in Balb/cmice, using saline and PEI injections as controls. PPE-EA and PEI weregiven at the same dose of positive charge (60 nmol of amino groups forPPE-EA and 60 nmol of total amino groups for PEI), to allow for a faircomparison with the assumption that the toxicity of these polymerspredominantly stems from their cationicity. As a result, PPE-EA wasgiven at a higher amount, 5.2 folds higher than PEI in mass (13.1 μg ofPPE-EA versus 2.5 μg of PEI per injection). Histological analysis at day7 revealed mild inflammatory reaction at muscle sites injected with thePPE-EA (FIG. 8), whereas severe inflammatory response was observed inthe PEI group. Moreover, severe necrosis was noticeable in all themuscle samples receiving PEI injection, with a large amount ofmacrophages, histiocytes and neutrophils present at the injection sites.

Example 8 Delivery of Interferon-α2b to Systemic Circulation UsingPPE-EA/DNA Complexes

A plasmid, pCMV-IVS-IFN-mod2, encoding interferon-α2b (IFN-α2b) was usedto test the effectiveness of PPE-EA as a carrier for the systemicdelivery of secretory proteins via intramuscular injection. It wasprovided by The Immune Response Corporation (Carlsbad, Calif.) as agift.

pCMV-IVS-IFN-mod2 plasmid DNA was dissolved in saline at a concentrationof 1.25 mg/ml. Complexes were prepared by adding 40 μl of PPE-EAsolution (0.41 mg/ml in saline) to 40 μl of DNA solution containing 50μg DNA to achieve the N/P ratio of 0.5 and vortexed for about 20seconds. The complexes were incubated at room temperature for 60minutes. Balb/c mice (6 to 8 weeks old, five to seven mice per group)received bilateral injections in the tibialis anterior muscle of 40 μlof complexes containing 25 μg of DNA. One group of mice receivedbilateral injections of 25 μg of plasmid DNA in 40 μl of saline, andanother group of mice receiving 40 μl of saline was used as thebackground control. The mice were bled at days 6, 10, 14 and 21, andserum samples were isolated and stored at −80° C. until assay. Theconcentrations of interferon-α2b in serum at different time points wereanalyzed using a human IFN-α ELISA kit (Pierce Endogen, Inc. Woburn,Mass.).

No significant level of IFN-α2b was detected in mice received 50 μg ofnaked DNA injection until day 14, reaching 917 pg/ml and 790 pg/ml ofIFN-α2b in serum on day 14 and 21, respectively. Comparing with nakedDNA injection, PPE-EA mediated gene transfer yielded a higher serumIFN-α2b of 1.61 ng/ml on day 14 (p<0.05), although IFN-α2b concentrationin serum declined to similar level as naked DNA on day 21 (FIG. 9).

1. A water soluble and positively charged biodegradable polymer that iscapable of forming a complex with negatively charged biologically activesubstances in aqueous solutions and comprises the recurring monomericunit shown in Formula I:

wherein R₁ is a divalent aliphatic organic moiety; R₂ is a positivelycharged alkyl or heteroalicyclic group selected from the groupsconsisting of primary amine, secondary amine, teriary amine, andquaternary amine; m is an integer from 1 to 6; n is from 20 to 2,000. 2.A water soluble and positively charged biodegradable polymer of claim 1,wherein the biodegradable polymer has between about 30 and about 200phosphate groups in the backbone.
 3. A water soluble and positivelycharged biodegradable polyphosphate of claim 1, wherein R₁ is defined inFormula II,

Wherein Each occurrence of R₃ and R₄ are independently selected from thegroup consisting of hydrogen or alkyl group; and q is 2 to
 4. 4. Amethod of preparing a water soluble and positively chargeablebiodegradable polymer of Formula I, comprising the steps of: (a)reacting a precursor polymer with recurring unit shown in Formula III,

wherein R₁ is a divalent aliphatic organic moiety; with an alcoholhaving a structure of HO(CH₂)_(m)R₂, wherein R₂ is a positively chargedalkyl or hertoalicyclic group selected from the groups consisting ofprotected primary amine, protected secondary amine, tertiary amine, andquaternary amine and m is an integer from 1 to 6; followed by (b)deprotecting the protected charge groups, if applicable.
 5. A method ofpreparing a water soluble and positively charged biodegradable polymeras described in claim 4, wherein the biodegradable polymer has between30 and 200 phosphate groups.